Products specific to pathogenic strains and their use as vaccines and in immunotherapy

ABSTRACT

The invention relates to isolated antigenic polypeptides obtainable by a process comprising the steps of: 1—selecting on the basis of sequence analysis those of the polypeptides which are either located in the outermembrane or secreted by the bacteria, 2—identifying the genes coding for said polypeptides which are conserved in B2/D clinical isolates, 3—purifying the polypeptides identified in step 1, which are found in step 2 to be conserved in B2/D isolates, 4—testing the polypeptides for immunogenicity using animals models. Application for making vaccines compositions and immunotherapies.

The invention relates to new products specific to pathogenic strains,particularly to extra-intestinal E. coli strains.

It more particularly relates as products to antigenic polypeptides andantibodies directed against said polypeptides and to their use asvaccines and in immunotherapy, respectively.

Although Escherichia coli is probably the best known bacterial speciesand is one of the most common isolated in clinical microbiologylaboratories, misconceptions abound regarding the various types of E.coli and the infections they cause.

E. coli strains of biological significance to humans can be broadlyclassified in 3 major groups:

1. Commensal strains, which are part of the normal flora.

2. Intestinal pathogenic strains, which are not part of the normalflora. This group contains various pathotypes (EPEC, EHEC, ETEC, EIEC)not including Shigella.

3. Extra-intestinal strains (ExPEC) which are responsible for infectionsoutside the gastro-intestinal (GI) tract, but can also be part of thenormal flora. All hosts, either immunocompromised or not are susceptibleto these infections.

ExPEC strains are responsible for the majority of the urinary tractinfections (UTI) particularly cystitis, pyelonephritis, and cathetherassociated infections.

They are also responsible for abdominal infections, nosocomialpneumoniae, neonatal meningitidis, soft tissue infections, and boneinfections. Each one of these localizations can lead to bacteremia witha risk of sepsis in case of organ failure.

ExPEC strains are indeed the most common Gram negative bacilli isolatedfrom blood cultures.

750 000 cases of bacterial sepsis occur each year in the US, and areresponsible for 225 000 deaths. In a recent study on 1690 cases ofsepsis, it was shown that the main bacteria species identified is ExPEC(16% of the cases) and then S. aureus (14% of the cases)

These numbers demonstrate the importance of ExPEC strains in bothhospital and community acquired infections.

ExPEC strains correspond to a homogenous subset of E. coli strains.Analysis of phylogenetic relationships among E. coli strains by MLEE hasrevealed that E. coli belong to 4 main phylogenetic groups designated A,B1, B2 and D.

The pathogenesis of ExPEC strains is that of extra-cellularmicroorganisms, i.e., they are well adapted to growth in theextra-cellular fluids and efficiently resist phagocytosis bypolymorphonuclear. Initial studies have shown that virulence factorsknown to be important for the extra-cellular growth are mainly found inB2/D E. coli., thus suggesting that B2/D subgroups contain most of theExPEC strains. This was reinforced by experiments performed on animalsshowing that B2/D strains are more virulent than A and B1 strains.Subsequent epidemiological studies have indeed confirmed thesehypotheses. B2/D isolates are those predominantly responsible forneonatal meningitidis (87%) and community or nosocomial acquiredurosepsis, (93% and 85%, respectively).

Similar results have been reported for cystitis (70% are due to the soleB2 E. coli), thus demonstrating that the importance of ExPEC strains.

These recent findings demonstrate that the B2/D subgroup of strains isthe E. coli core genome the best adapted to growth in extra-cellularfluids.

In addition to this core genome, ExPEC strains have variouspathogenicity islands which encode virulence factors associated with thedifferent pathogenesis of extra-intestinal E. coli infections (UTI,urosepsis, neonatal meningitidis . . . ). Among the main virulencefactors are the capsule, which is well-known to be important forextra-cellular growth, and the iron chelation systems (aerobactin andenterochelin, for example). In addition, depending on the pathogenesis,these strains can produce toxins (CNF, hemolysin . . . ), adhesins (pap,sfa . . . ) and other iron chelation systems.

The notion that B2/D E. coli correspond to a distinct subset ofpathogenic E. coli strains is reinforced by the fact that B2/D E. coliare not broadly isolated from the stools of humans. They were recoveredfrom only 11% of individuals, whereas A and B1 subgroups are present inthe stools of 74% of the individuals of a human population.

As mentioned above the pathogenesis of ExPEC strains relies on theirability to multiply in the extra-cellular fluids and to resistbactericidal activity of the complement and phagocytosis bypolymorphonuclear. Therefore, as for other extra-cellular pathogens(Haemophilus influenzae, Streptococcus pneumonieae and Neisseriameningitidis) a protective antigen against ExPEC has to induceantibodies that promote opsonisation and/or the bactericidal activity ofserum.

Considering the above statements, an efficient antigen has to be largelyrepresented among the population of B2/D E. coli. Similarly to otherextra-cellular pathogens, the capsular polysaccharide would be an idealantigen, however most pathogenic B2 strains express the K1polysaccharide. The latter has a structure identical to that of group Bmeningococcus, which is non-immunogenic and shares common antigens withthe brain. Another possible target may be the lipopolysaccharide (LPS).However there are a large number of different LPS serotypes that areshared by various subgroups.

The inventors have now found that some specific components coded by theB2/D genome, but absent from A and B1 E. coli strains, are particularlyuseful as antigens and can specifically prevent the pathologies due toExPEC strains. Homologs of these antigenic components can be found inother pathogenic bacterial species and therefore are useful to preventthe pathologies caused by these bacteria. Accordingly, any reference toproducts specific to ExPEC strains and to their uses will encompasscomponents in these species.

For example homologuous antigens could be present in the followingspecies and be as such used for prevention of disease due to thebacteria:

Pseudomonas aeruginosa, Escherichia coli O157:H7, Yersinia pestis,Vibrio cholerae, Legionella pneumophila, Salmonella enterica, Salmonellatyphimurium, Haemophilus influenzae, Neisseria meningitidis, Neisseriagonorrhoeae, Bacillus anthracis, Burkholderia cepacia, Campylobacterjejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Clostridiumbotulinum, Clostridium difficile, Cryptococcus neoformans, Enterobactercloacae, Enterococcus faecalis, Helicobacter pylori, Klebsiellapneumoniae, Mycobacterium leprae, Mycobacterium tuberculosis,Pseudomonas aeruginosa, Salmonella paratyphi, Salmonella typhi,Staphylococcus aureus, Klebsiella pneumoniae, Listeria monocytogenes,Moxarella catarrhalis, Shigella dysenteriae, Shigella flexneri, Shigellasonnei, Staphylococcus epidermidis, Streptococcus pneumoniae, and anyspecies falling within the genera of any of the above species.

It is then an object of the invention to provide new isolated antigenicpolypeptides, and polynucleotides belonging to the core B2/D genome andnot present in commensal E. coli.

Another object of the invention is to provide antibodies raised againstsuch antigenic polypeptides, or peptidic fragments.

It is still another object of the invention to provide vectors and hostcells containing said polynucleotides.

Another object of the invention is to provide vaccine compositionsspecific to extra intestinal infections caused by ExPEC and pathologiescaused by other pathogenic strains expressing antigenic polypeptideshomologous to the ExPEC antigenic polypeptides.

The invention also relates to means for detecting and treating adevelopment of E. coli in a human or animal compartment which isextra-intestinal (systemic and non-diarrhoeal infections, such assepticaemia, pyelonephritis, or meningitis in the newborn).

The isolated antigenic polypeptides used according to the invention areselected among polypeptides specific to B2/D E. coli strains and notpresent in A and B1 isolates of E. coli. They are encoded by genesbelonging to the core B2/D genome and are not present in commensal E.coli.

They have a sequence selected in the group comprising the sequences ofSEQ ID No11 to No66 or 133-145 or homologous sequences with a minimum of25% of identity with the whole sequences SEQ ID No11 to No66, or133-145, respectively.

The isolated polypeptides having SEQ ID No 14, 15, 17, 21, 22, 23, 28,29, 30, 32, 36, 38, 39, 41-44, 46, 49, 50, 52 to 55, 58, 60, 63, 133-138are new polypeptides and therefore are part of the invention.

The invention also relates to homologous isolated antigenic peptides,comprising polypeptides having at least 25% identity to a polypeptidehaving a sequence SEQ ID No as above defined, more particularly havingSEQ ID No14, 15, 17, 21, 22, 23, 28, 29, 30, 32, 36, 38, 39, 41-44, 46,49, 50, 52 to 55, 58, 60, 63, 133-138, or at least 25% identity to afragment comprising at least 5, at least 10, at least 20, at least 30,at least 40, at least 50, at least 60 or more than 60 consecutive aminoacids of a polypeptide having a sequence corresponding to said SEQ IDNos, as determined using BLASTP or BLASTX with the default parameters.

Said polypeptides are obtainable by a process comprising the steps of:

-   -   a—selecting on the basis of sequence analysis those of the        polypeptides which are either located in the outermembrane or        secreted by the bacteria,    -   b—identifying the genes coding for said polypeptides which are        conserved in B2/D clinical isolates,    -   c—purifying the polypeptides identified in step a, which are        found in step 2 to be conserved in the B2/D isolates,    -   d—testing the polypeptides for immunogenicity using animals        models.

By the term “conserved”, it is meant, according to the invention, thatthe genes coding for the polypeptides are present with a frequency of atleast 50% in B2/D isolates, preferably greater than 60%, more preferablygreater than 80% and even more preferably greater than 85%, and in lessthan 40% in A/B isolates; preferably in less than 20%, more preferablyin less than 15%.

The animal models used in step c are infected adult animals, eventuallyimmunodepressed.

The adult animals particularly mice, are infected intraperitoneally, theendpoint being the animal death and/or bacteremia measurement.

The animals can be immunodepressed by injection, for example, ofcyclophosphamide which induces a neutropenia. Such a model will validatethe use of the antigen for prevention of E. coli sepsis inimmunodepressed patients. Another animal model could be for example 2 to3 day old infant mice.

The variants or fractionnal sequences conserving the B2/D properties andwhich are antigenic as defined in step 4 of the above process are alsopart of the invention. The term “variant” is herein intended to mean anysequence having insertions and/or deletions and/or substitutions withrespect to the parent sequence. The term “fractional” is herein intendedto mean any fragment of the parent sequence.

The invention also relates to the use of isolated polynucleotides codingfor a polypeptide such as above defined according to the universalgenetic code and taking into account the degeneracy of this code. Theterm “polynucleotide” encompasses any nucleotidic sequence such as DNA,including cDNA, RNA, including mRNA.

Said polynucleotides have preferably sequences corresponding to SEQ IDNo77 to SEQ ID No132 or 146 to 158

More preferably, said polynucleotides have sequences corresponding toSEQ ID No 80, 81, 83, 87, 88, 89, 94, 95, 96, 98, 102, 104, 105,107-110, 112, 115, 116, 118, 119, 126, 127, 130, 132, 135, 146-151.

The invention also relates to the homologs to said polynucleotides. Saidhomologs may have at least 25% identity to a polynucleotide having saidsequences, or at least 25% identity to a fragment comprising at least15, at least 30, at least 60, at least 90, at least 120, at least 150,at least 180 or more than 180 consecutive nucleotide of a polynucleotidehaving one of said SEQ ID Nos, as determined using BLASTN with thedefault parameters, and are encompassed by the invention inasmuch asthey are capable of coding for a polypeptide having the antigenicproperties of those according to the invention.

The present application is also aimed towards any vector comprising atleast one of said polynucleotides and also any cell transformed bygenetic engineering, characterized in that it comprises, bytransfection, at least one of said polynucleotides and/or at least onevector according to the invention, and/or in that said transformationinduces the production by this cell of at least one polypeptidecorresponding to a polynucleotide such as above-defined.

The invention also relates to a process for isolating and identifyingantigenic polypeptides, therefore useful as vaccine for E. coli.

Such a process comprises the steps of

-   -   a—selecting on the basis of sequence analysis those of the        polypeptides which are either located in the outermembrane or        secreted by the bacteria,    -   b—identifying the genes coding for said polypeptides which are        conserved in B2/D clinical isolates,    -   c—purifying the polypeptides identified in step a, which are        found in step 2 to be conserved in B2/D isolates,    -   d—testing the polypeptides for immunogenicity using animals        models.

The selected antigenic polypeptides, alone or in combination, arecapable of inducing an antibody response for prevention of infectionsdue to ExPEC strains regardless of the pathogenesis and of the infectionsite (UTI, pyelonephritis, sepsis, bacteremia, neonatal meningitis).

Such polypeptides particularly have sequences SEQ ID No1 to SEQ ID No66,or 133-145 or correspond to homologous sequences.

The invention thus relates to vaccine compositions specific to E. coliextra-intestinal infections, comprising an effective amount of at leastone antigenic polypeptide or fragment thereof as above defined, with acarrier, particularly at least one polypeptide of SEQ ID No1 to SEQ IDNo66, except SEQ ID No8, and 133-145 and the homologous polypeptides.

Such vaccine compositions are particularly useful for preventing urinarysystem infections, pyelonephritis, sepsis, bacteremia, neonatalmeningitis.

The vaccine compositions of the invention are indicated for

-   -   immunodepressed patients, ideally before the start of the        immunosuppressive therapy: patients suffering from cancer,        diabetes, leukaemia, transplant patients, patients receiving        long-term steroids therapy.    -   Patients before surgery where there is a high risk of E. coli        infections (abdominal surgery).    -   In all these cases, the E. coli vaccine of the invention could        be administered in association with a Staphylococcus aureus        vaccine,    -   Patients with recurrent UTI, especially after one episode of        pyelonephritis.    -   The prevention of neonatal infections will require vaccination        of the mother, implying vaccination long before pregnancy to        avoid potential problem. Ideally such a vaccine should be        associated with a Group B Streptococcus polysaccharide vaccine        in order to also prevent late onset neonatal infections. It        should be pointed out that the induction of a level of        antibodies against B2/D E. coli in pregnant women would also        prevent UTI, which are always a risk in the context of a        pregnancy.        The formulation and the dose of said vaccine compositions can be        developped and adjusted by those skilled in the art as a        function of the indication targeted, of the method of        administration desired, and of the patient under consideration        (age, weight).

These compositions comprise one or more physiologically inert vehicles,and in particular any excipient suitable for the formulation and/or forthe method of administration desired.

For example the vaccine could be a suspension of the purifiedpolypeptide in sterile water with aluminium based miniral salt asadjuvant and be administred subcutanously with a first and boostinginjection.

The antibodies raised against the above-identified polypeptides are alsopart of the invention.

They are capable of binding to said polypeptides in physiological-typeconditions (in vivo or mimicking in vivo) when administered to a humanor animal organism, and ELISA-type conditions when said binding productis intended to be used in assays and methods in vitro. Such antibodiesadvantageously inhibit the extra-intestinal growth of ExPEX strains inhuman or animal.

They are particularly useful for immunotherapy applications withantibodies specific to polypeptidic antigens, for treatment andprevention of severe infections in at risk populations such as neonatesor patients undergoing surgical procedures. For these applicationsspecific human monoclonal antibody (Mab) will be derived from thepeptides or polypeptides.

The methods for manufacturing such antibodies using the polypeptidesaccording to the invention are available to those skilled in the art.They are conventional methods which comprise, in particular, theimmunization of animals such as rabbits and the harvesting of the serumproduced, followed optionally by the purification of the serum obtained.A technique suitable for the production of monoclonal antibodies is thatof Köler and Milstein (Nature 1975, 256:495-497).

Said antibodies do not recognize the cells of the human or animal towhich it is intended.

In particular for immunotherapy applications with monoclonal antibodiesspecific to polypeptidic antigens, for treatment and prevention ofsevere infections in at risk populations such as neonates or patientsundergoing surgical procedures. For these applications specific humanmonoclonal antibody will be derived from the peptides or polypeptides.

The antibodies or fragments thereof are advantageoulsy humanized whenintended for a human administration.

Alternatively, humanized Mab could be derived from murine or rat Mabspecific of the antigen. These fully humanized Mab are constructed usingconventional molecular techniques to graft complementarity-determiningregions from the parent murine or rat antibacterial antibody into humanIgG1 kappa heavy and light-chain frameworks.

The present invention is also aimed towards the use, in an effectiveamount, of at least one of polypeptides having SEQ ID No14, 15, 17, 21,22, 23, 28, 29, 30, 32, 36, 38, 39, 41-44, 46, 49, 50, 52 to 55, 58, 60,63, 133-138, antibodies or polynucleotides for the diagnosis of thepresence or absence of undesirable extra-intestinal E. coli, and/or forthe diagnosis of an extra-intestinal E. coli infection.

The detection of the presence or absence of such compounds can inparticular be carried out by nucleotide hybridization, by PCRamplification or by detection of their polypeptide products. Detectionof the presence of such compounds makes it possible to conclude that aB2/D E. coli strain is present.

The invention also relates to pharmaceutical compositions foralleviating and/or preventing and/or treating an undesirable growth ofE. coli comprising an effective amount of at least one polypeptide asabove defined, particularly having SEQ ID No1-66 to 133-145, incombination with a pharmaceutically acceptable carrier.

Preferred pharmaceutical compositions comprise at least one polypeptidehaving SEQ ID No14, 15, 17, 21, 22, 23, 28, 29, 30, 32, 36, 38, 39,41-44, 46, 49, 50, 52 to 55, 58, 60, 63, 133-138,

The present application is also aimed towards any use of a polypeptidesuch as above defined for the manufacture of a composition, inparticular of a pharmaceutical composition, intended to alleviate and/orto prevent and/or to treat an undesirable growth of E. coli, such as anE. coli infection, (for example systemic and non-diarrhoeal infections),the presence of extra-intestinal E. coli or a sanitary contamination.

The present invention is illustrated by the examples which follow andwhich are given in a non limiting capacity and with reference to FIGS. 1and 2, wherein

FIG. 1 represents a protein purification result after cloning andexpression, and

FIG. 2 is a picture of the DNA array after hybridization with thegenomic DNA from a B2/D reference strain.

EXAMPLE 1 Assay for the immunogenicity of a selected polypeptide fromsequences 1-66 and 133-145 (except SEQ ID No8)

Cloning Expression and Purification of the Selected Polypeptide.

The nucleic acid having SEQ ID No95 encoding the polypeptidecorresponding to SEQ ID No28 was cloned without the signal sequence(coding the 16 first amino acids) in a prokaryotic expression vectoraccording to classical methods for cloning. The recombinant plasmid wasused to transform the E. coli strain BL21. Transformed cells containingthe recombinant plasmid were selected in LB medium with 100 μg/mlampicillin. Individual clones are picked and grown in presence of IPTG 1mM to induce recombinant protein expression. Total protein content ofthe culture cells was extracted by cell lysis. Recombinant protein waspurified by affinity columns.

Protein Purification after Cloning and Expression

Total cell lysat of IPTG-induced bacteria were mixed with Ni-NTA matrix(Qiagen®) for 60 min et 4° C. and loaded into a column. After washingthe column to remove non specific binding, the recombinant protein waseluate 3 times with 1 ml elution buffer pH 5.9. The protein was theneluate 4 times with 1 ml elution buffer pH 4.5.

FIG. 1 represents a Coomassie blue stained SDS gel of recombinantprotein after affinity column purification: PM: markers E1-4: samplecollected from each purification fraction. Arrow indicate the bandcorresponding to the recombinant protein.

Test for Immunogenicity in an Animal Model

Polypeptide preparation from SEQ ID No28 was injected to Swiss mice toinduce an antibody response as follows:

At d0 a first immunisation was done by injecting 20 μg of the protein atin 100 μg solution of PBS and complet Freund adjuvant (1:1). Controlanimals were injected with 1001 solution of PBS and complet Freundadjuvant (1:1).

Boosting injection at d21 with 10 μg of protein in 100 μl PBS andcomplet Freund adjuvant (1:1).

Sera from vaccinated animals was prepared from blood drawn by puncturein the tail of the mice.

Detection of specific antibodies in animal sera, at d20 before theboosting injection, was performed by western blot according to standartprotocol. Purified polypeptide was subjected to electrophoresis (10 μgper lane) and transfert to nitrocellulose membrane.

The membranes were then saturated by incubation 35 min with PBS/Tween200.1%/powder milk 5%.

Diluted sera was incubated with the membrane for 45 min. Membranes werewashed three time 5 min with PBS/tween. Bound antibodies were thenrecognized by an anti-mouse IgG coupled to horseradish peroxidaseenzyme. After washing 3 times with PBS/Tween and 3 time with PBS,enzymatic activity was revealed by addition of chromogenic substrate DABand hydrogen peroxyde.

Results: Sera from vaccinated animal, diluted at 1/100 revealed a uniqueband corresponding to the injected polypeptide. No antibody to thepolypeptide could be detected in sera from control animals.

At d42, 300 μl of cyclophosphamide and 200 μl at d45 were injected IP inthe mice to induce neutropenia in order to increase the susceptibilityto the challenge infection.

At d46 vaccinated and control mice were challenged by intraperitonealinjection of the wt B2/D strain C5 of E. coli at a dose equal to 10 timethe LD50 (letal dose)

Immunogenicity of the selected polypeptide and protection conferred byvaccination with the seleted polypeptide was assessed by the survival ofvaccinated animals three days post challenge.

Another example of vaccination to demonstrate immunogenicity ofpolypeptides:

-   -   24 Balb/c mice, female, 6 weeks old were immunized by        subcutaneous Injection of the protein in Complete Freund's        adjuvant, and 14 control mice were injected with CFA and PBS 3        weeks later a boost injection of 10 μg of protein in with        incomplete Freund's adjuvant.

Before challenge at d62, sera was collected at d61 to analyze theantibody response in the vaccinated animals.

-   -   WB analysis of sera from immunized mice were performed to detect        the antibody response to the recombinant proteine used for        immunization as described above.    -   An ELISA assay was set up to measure antibody titer in        vaccinated animals:    -   Each recombinant protein was coated on a 96-well plates with        (200 ng/100 μl coating buffer), plates are saturat with 3% BSA        in PBS.    -   Twofold serial dilution of sera were made in PBS 1×/1% BSA from        1:500, to 1:1024000 and incubated on the plate, antiboby binding        was reavealed using a rabbit Anti-mouse IgG conjugated with        Peroxydase and OPD(o-phenylenediamine) chromogen substrate.    -   Read the OD₄₉₅ with Sanofi Diagnostics Pasteur PR2100®

Results: TABLE 1 Sera titer against recombinant protein by ELISArecombinant SEQ ID serum1 serum 2 serum 3 serum 4 Control protein 2 12864 128 512 <0.5 140 >64 >64 >64 >64 <0.5 + 31 >64 >64 >64 >64 <0.5 +49 >64 >64 >64 >64 <0.5 + 51 >64 >64 >64 >64 <0.5 + 25 >64 >64 >64 >64<0.5 + 7 16 8 >64 <0.5 <0.5 + 19 >64 >64 >64 >64 0.5 + 3 >64 >64 >64 >64<0.5 + 26 512 128 64 256 <0.5 + 18 >64 >64 >64 >64 0.5 +32 >64 >64 >64 >64 0.5 + 53 >64 >64 >64 >64 0.5 + 587 >64 16 32 32<0.5 + 11 >64 32 32 64 <0.5 + 36 512 256 512 256 <0.5 + 10 32 128 256128 0.5 + 47 512 512 512 512 0.5 + 20 1024 256 256 512 <0.5 + 17 1024512 128 512 <0.5 +

-   -   To assess the ability of sera to recognize the native antigen        expressed by the bacteria, western blot was also performed on        whole bacteria lysat.

To this end, bacteria were grown in LB medium supplemented or not withiron chelator until OD600=0.5-0.6 and pelleted by centrifugation 5minutes at 10000 rpm. The pellet was lysed by resuspension in 1× loadingbuffer containing SDS and heated 5 min at 95° C. before migration on thegel. Western blot assay was the performed with sera from controls andvaccinated animals.

Results in table 2 shows the results obtained with Sera from vaccinatedmice against recombinant protein and against E. coli lysat. TABLE 2reactivity in Western Blot of sera from mice vaccinated withpolypeptides encoded by the different ORFs whole cell recombinant SEQ IDN° lysate protein 2 + + 140 + + 26 + +

-   -   Protection assay, end point: mortality

At d62, 20 vaccinated and 10 control mice were challenged with an E.coli virulent strain belonging to B2 group at a dose equal to the LD 50(5.10⁵ cfu/mice) by intraperitoneal injection. Mortality is recorded at48 h, results in Table 3 are expressed as a percentage of protectionrepresenting the difference of survival in vaccinated versus controlmice goups. TABLE 3 Protection obtained in mice challenged afterimmunization with proteines encoded by the corresponding ORFs. SEQ ID N°% protection 2 52 26 66 36 46 10 30 47 60 20 25

-   -   Protection assay, end point: bacteremia

At d62, 10 vaccinated and 5 control mice were challenged with an E colivirulent strain belonging to B2 group at a dose equal to the ⅕ of the LD50 (1.10⁵ cfu/mice) by intraperitoneal injection. With this infectiousdose the mice survived the infection at d48. At 48 h blood was collectedfor each mice in presence of heparin. To assess bacteremia, the bloodwas plated on LB media and colonies count measured after overnightculture.

EXAMPLE 2 Distribution of the DNA Sequence of ORFs Specific for B2/DGroup of E. coli in Clinical Isolates

To make a DNA arrays membrane specific for B2/D group of E. coli, DNAcorresponding to ORFs that were identified as specific for B2/D coregenome of E. coli was amplified by PCR and spotted on nylon membraneusing standard methods to those skilled in the art.

Chromosomal DNA from 30 E. coli clinical isolate strains (of which 23were from pathological conditions and 6 isolated from human normalflora), was prepared and radiolabelled with 33P.

DNA from these clinical isolates was then hybridized to the B2/Dspecific DNA array, the results were read by a phosphoimager and spotsreactivity was analyzed with an image analysis software. Ifhybridization gave a positive signal for a particular ORF, this ORf isconsidered to be present in the genome of the isolate. Quality controlof the array is the hybridization of a probe DNA from a reference strainof E. coli as shown in FIG. 2, which represents a picture of the DNAarray after hybridization with the genomic DNA from a B2/D referencestrain.

The details of the method used for these experiments has been describedpreviously in Tinsley et al. Methods Enzymol. 2002, 358; 188-207.

Results presented in Table 4 are expressed as the frequency of each ORFdetection in the three different group A, B and D of E. coli strains.TABLE 4 Presence of Orfs encoding antigens in E. coli clinical isolategenomes Frequency of Clinical isolate groups positive signal % A D B2SEQ ID N° N = 6 n = 5 n = 18 86 17 20 100 119 0 80 100 137 0 0 100 77 0100 94 78 0 100 100 79 0 100 100 80 0 0 100 84 0 0 100 82 0 0 100 88 0 0100 83 0 0 94 85 0 0 78 88 0 0 56 81 33 20 100 89 33 60 67 90 0 0 61 9117 80 67 94 0 0 100 92 0 0 100 93 0 0 100 96 0 100 100 85 17 100 100 970 70 100 98 0 0 100 99 0 0 78 101 0 80 100 102 0 0 100 104 0 0 11 103 00 100 105 0 0 67 100 0 100 100 111 17 0 61 115 0 0 78 114 0 0 83 113 0 094 120 33 80 16 125 17 0 89 109 0 0 33 110 0 0 17 124 0 0 72 126 0 80 78116 0 0 33 112 0 0 22 106 17 0 33 117 33 80 11 132 33 80 22 122 0 0 2274 0 0 89 70 0 0 33 73 0 0 89 71 50 100 22 75 0 0 0 76 0 0 56 67 0 0 5069 100 100 100 68 67 100 100 152 0 0 94 153 0 100 100 150 0 0 11 142 83100 78 157 67 0 94 156 17 100 100 100 0 100 100 154 0 80 67 147 0 0 100146 17 100 100 158 17 100 89 107 17 0 78 72 50 100 44 151 0 0 11 149 0 028 148 0 0 6

EXAMPLE 3 Vaccines Compositions Intended for Prevention of Form ofInfection by ExPEC

The polypeptide coded by a sequence comprising SEQ ID No28 is conjugatedwith a toxin and added to a physiologically inert vehicle.

This conjugated peptide is optionnally added to a childhood vaccine.

The composition is sterilized and can be injected parenterally,subcutaneously or intramuscularly.

Said composition can also be sprayed onto mucosa with the aid of aspray.

1. The isolated polypeptides having SEQ ID NO0; 14, 15, 17, 21, 22, 23,28, 29, 30, 32, 36, 38, 39, 41-44, 46, 49, 50, 52 to 55, 58, 60, 63,133-138.
 2. Isolated antigenic polypeptides according to claim 1obtainable by a process comprising the steps of: a—selecting on thebasis of sequence analysis those of the polypeptides which are eitherlocated in the outermembrane or secreted by the bacteria, b—identifyingthe genes coding for said polypeptides which are conserved in B2/Dclinical isolates, c—purifying the polypeptides identified in step a,which are found in step b to be conserved in B2/D isolates, d—testingthe polypeptides for immunogenicity using animals models.
 3. Isolatedpolynucleotides, coding for a polypeptide according to claim 1,according to the universal genetic code.
 4. Isolated polynucleotidesaccording to claim 3, having sequences selected in the group comprisingSEQ ID NO80; 81, 83, 87-89, 94-96, 98, 102, 104, 105,107-110, 112, 115,116, 118, 119, 126, 127, 130, 132, 135,146-151.
 5. An expression vectorcomprising at least an isolated polynucleotide according to claim
 3. 6.A host cell comprising an expression vector according to claim
 5. 7. Aprocess for isolating and identifying antigenic polypeptides, useful asvaccines comprising the steps of: a—selecting on the basis of sequenceanalysis those of the polypeptides which are either located in theoutermembrane or secreted by the bacteria, b—identifying the genescoding for said polypeptides which are conserved in B2/D clinicalisolates, c—purifying the polypeptides identified in step a, which arefound in step b to be conserved in B2/D isolates, d—testing thepolypeptides for immunogenicity using animals models.
 8. The process ofclaim 7, comprising the use of infected adult animals, eventuallyimmunodepressed, and of infant animals as models for neonatalinfections.
 9. The use of at least one polypeptide or fragment of thesepolypeptides selected in the group comprising SEQ ID NO1 to SEQ ID NO66(except SEQ ID NO8), or 133-145 as antigens and the homologoussequences.
 10. A vaccine composition specific to E. coliextra-intestinal infections, comprising an effective amount of at leastone antigenic polypeptide such as selected by the process of claim 7,alone or in combination, particularly at least one polypeptide having asequence selected in the group comprising SEQ ID NO1 to SEQ ID NO66, or133-145, except SEQ ID NO8 and the homologous sequences, with a carrier.11. The vaccine composition of claim 10 for preventing urinary systeminfections, pyelonephritis, sepsis, bacteremia, neonatal meningitidis.12. The vaccine composition of claim 10, adapted to specific indicationin combination with components directed against other bacteria, such asS. aureus or group B Streptococcus. Or other bacteria implicated insystemic infections.
 13. Antibodies or fragments thereof directedagainst a polypeptide such as used according to claim
 9. 14. Monoclonalantibodies against epitopes of polypeptide and there use aspharmaceutical compound for treatment or prevention of severe infectiondue to Expec in neonates and patients at risk for such infections.
 15. Amethod for detecting the presence or absence of undesirableextra-intestinal E. coli, and/or for the diagnosis of anextra-intestinal E. coli infection, comprising the use of at least onepolypeptide such as defined in claim 1 or a polynucleotide encoding thesame, or an antibody neactive thereto to, said polypeptide(s) beingoptionally in combination with anyone of the polypeptides having SEQ IDNO1-66 to 133-145.
 16. Pharmaceutical composition for alleviating and/orpreventing and/or treating an undesirable growth of E. coli comprisingan effective amount of at least one polypeptide according to claim 9, incombination with a pharmaceutically acceptable carrier.